Recombinant PNGase F

Cat. No.: R488845

GRADE & PURITY Specific Activity ＞25 U/mg;Activity 5 U/ml

Bioactivity

5 U/ml

Overview

Product Description

Recombinant PNGase F is isolated from a E. coli strain containing a clone of the Elizabethkingia miricola gene. There is no detectable difference in activity or specific activity of the recombinant enzyme from the native enzyme.

PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.

Contents

60 µl aliquot of recombinant PNGase F (0.3 U) in 20 mM Tris-HCl, pH 7.5
Included with 20 µL and 60 µL pack sizes
5x PNGase F Reaction Buffer 7.5 for PNGase F – 250 mM sodium phosphate, pH 7.5
PNGase F Denaturation Solution – 2% SDS, 1 M Beta-mercaptoethanol
PNGase F Triton X-100 – 15% solution

Formulation

The enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl (pH 7.5).

Molecular Weigh

approximately 35 kD.

pH optimum

7.5, active over the range 6-10.

Specific Activity

Specific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of RNase B in 1 minute at 37˚C, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).

Specificity

PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A. 

Stability

Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. 

Quality & Purity

PNGase F is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.

PNGase F Suggested usage

1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 35 µl final volume with de-ionized water.

2. Add 10 µl 5x PNGase F Reaction Buffer 7.5 and 2.5 µl of PNGase F Denaturation Solution. Heat at 100˚C for 5 minutes.

3. Cool. Add 2.5 µl of PNGase F Triton X-100 and mix.

4. Add 2.0 µl of PNGase F to the reaction. Incubate 3 hours at 37˚C.

If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours. Monitor cleavage by SDS-PAGE.

Specifications

Product Name

Recombinant PNGase F

Synonyms

Peptide-N4-(acetyl-ß-glucosaminyl)-asparagine amidase, N-Glycosidase F

Specifications & Purity

Specific Activity ＞25 U/mg;Activity 5 U/ml

Bioactivity

5 U/ml

Enzyme Commission Number

3.5.1.52

Molecule Type

Protein

Storage and Shipping

Concentration

Specific Activity ＞25 U/mg;Activity 5 U/ml

Storage

Store at 2-8°C

Shipped In

Wet ice